ABSTRACT
Objectives:
The effects of a composite nanofiber wound dressing material consisting of a polyvinylidene alcohol and polyvinylidene pyrrolidone polymer mixture with a hemostatic agent doped with Ankaferd Blood Stopper (ABS) on the healing of experimentally induced dermal wounds in rats were examined.
Conclusion:
It was concluded that the ABS-dopped dressing did not have a negative effect on wound healing, it accelerated healing, and it could be used effectively and safely to treat skin injuries. However, further studies are needed to evaluate the clinical and histopathological benefits and potential adverse effects of wound dressings produced using ABS-doped polymers on wound healing.
Results:
In the histopathological examinations, macroscopic evaluations revealed a statistically significant difference between the groups in terms of wound diameter, reepithelialization, and inflammation formation (p= 0.019). In parallel with wound healing and histological outcomes, TAS values increased in the ABS-doped groups, and TOS and OSI values decreased in the wound dressing groups (p < 0.05).
Materials and Methods:
Rats were divided into 4 groups (n= 6). Histological material was examined on tissues taken from the wound site, whereas total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) analyses were performed on blood samples taken from the cardia. The material that was produced had hydrophilic properties, and both the ABS-doped and-undoped forms of the material positively affected wound healing.
INTRODUCTION
A skin wound is an injury that compromises the integrity of the epidermis as a physical barrier, thus disrupting its normal anatomical composition and physiology.1 Today, millions of people are injured due to various causes, and many hemostatic agents are used to stop bleeding, with different levels of action and duration of stopping bleeding. The Ankaferd Blood Stopper (ABS), which is used after surgery and is effective in many cases such as bleeding gums, is an antibacterial hemostatic agent without additives consisting of various ratios of dried roots and leaves of Thymus vulgaris L., Glycyrrhiza glabra L., Vitis vinifera L., Alpinia officinarum Hance, and Urtica diocia L., which are plants that are used in traditional Turkish medicine.2,3,4
In the intermediate recovery step, hemostasis is a natural process consisting of the inflammation, proliferation, and maturation stages.5,6 In this process, the general characteristics of the injured tissue should be well known, and tissue-specific treatment methods should be applied. The aim of these methods is to create an ideal environment for epithelial formation in the injured area by stimulating the inflammatory cells, platelets, and extracellular matrix involved in wound healing.5 Wound dressings are among the treatment methods that can create an ideal environment for proliferation by mimicking the extracellular matrix, protect the wound against microorganisms and infections, and contribute to the healing process.
Today, with the discovery of new-generation biopolymers and the development of novel production techniques, modern wound dressings are being produced as an alternative to conventional wound dressings.7 Polyvinyl alcohol (PVA) and polyvinylidene pyrrolidone (PVP), which are non-toxic, bio-based, renewable, and sustainable polymers, are used in biomedical, pharmaceutical, and regenerative medicine.8 They can be converted into biocompatible and biodegradable, water-retaining, water-soluble, and nanofiber forms.9 As natural synthetic polymers, PVA and PVP are easy to process because they have controllable physical properties and mechanical strength, and so, they are widely used for wound dressings.10,11,12 Electrospinning, which is a nanofiber production technique, is a preferred method aimed at producing nanodiameter fibers by the electric field effect of polymer-based gels, and it is preferred because fibers that are one hundred times smaller (with an average radius of 10 nm-500 nm) can be produced compared to those produced by the classical method.13,14,15 It is stated that the products used in treatments with experimental practices reduce oxidative stress (TOS/TAS = OSI) by increasing the total antioxidant status (TAS).16,17 OSI analysis is used to determine oxidative stress levels as an easy, precise, automated, and inexpensive method.
Although various polymers and methods are used in nanofiber production, there is no study in the literature in which ABS-doped PVA and PVP polymers were produced by the electrowinning method. The aim of this study was to investigate the healing process and biochemical effects of a medical textile product containing an ABS-added polymer that can stop bleeding and heal wounds quickly in a rat model with experimentally induced wounds. Scanning electron microscopy (SEM) and Fourier transform infrared spectrophotometry (FTIR) analyses were carried out to characterize ABS dopped and non-dopped ABS polymers in the production of the material.
MATERIALS AND METHODS
The study was initiated by obtaining the approval of the ethics committee of Selçuk University, Experimental Medicine Research and Application Center (decision date: 25.01.2019 and decision number: 2019-2). All chemical materials used in the study were supplied by Sigma and Merck.
RESULTS
DISCUSSION
Injuries caused by traumas, surgical operations, and burns can lead to serious health problems, ranging from disability to death. Wound dressing is a practical process that allows the wound to heal in a short time under hygienic conditions.38 If bleeding occurs in the injured area, wound dressings containing hemostatic additive agents may be preferred.39 In this study, the effects of wound dressings containing nanofibers with doping with ABS, which is a hemostatic agent, on wound healing were evaluated in macroscopic, histopathological, and biochemical terms.
Hydrophilicity is a sought-after feature for wound dressing materials.35,36,40,41,42 PVA and PVP are biocompatible polymers with biodegradability and non-toxic properties9. The addition of silver nanoparticles to PVA and PVP had a notable effect on wound healing in New Zealand white rabbits.14 It was determined that the wound dressing used in this study was sufficient to provide the permeability of fiber diameters. Moreover, no droplet or lump-like formation was observed on the surface. Because of these properties, it is thought that the substance produced in this study would be suitable for use and may be a preferred method in case of injuries.
During the recovery process, wound diameter was examined for reepithelialization and inflammation formation. In the measurements made on the last day of the study, the mean wound diameter in the ABS-dopped wound dressing group was much smaller than that in the control group, whereas the mean wound diameter in the local ABS-treated group was higher than that in the control group. This situation was interpreted as the ABS-dopped wound dressing being effective in healing, and the local ABS application slowed down healing.
There are many studies in the relevant literature investigating the effects of ABS on wound healing. It was reported that ABS is an agent that accelerates recovery and is effective in accelerating the wound healing process in periodontal treatment.43 Similarly, ABS is effective in trauma-related soft tissue defects that cannot be repaired by primary closure.44 ABS was observed to accelerate the healing process in bone defects created in the rat tibia.45 It was shown that ABS applied to wounds on the back skin of rats accelerated healing.46 According to a similar study, ABS speeds up wound healing in rats during the early period.47 ABS has also been reported to be effective in healing full-thickness skin wounds in rats.39 It was seen to provide healing even in second-degree burns.4 In a similar study, ABS had a positive effect on wound healing in rats.27 It was also stated that ABS has a positive effect on wound healing in diabetic rats.48 In contrast with the aforementioned results, ABS was not found to be effective in preventing postoperative intra-abdominal adhesions, and its use could be harmful because it caused abdominal organ damage.49 In the same study, it was stated that ABS did not prevent adhesions but increased them on a macroscopic level.
Our findings supported previous results, and the ABS-dopped dressings in this study were effective in reducing the wound size from 15x15 mm to 1x1 mm, thus contributing to healing. Although our results were similar to those of other studies in the literature in which the positive effects of ABS on wound healing have been stated,26,27 they differed from some others showing that ABS is not effective alone, and it even slows healing. It was thought that the amount of ABS used in each study, the diameter of the wound that is created, and the age and sex of the animals may be different. The cycle occurring in females may have affected the results of our study by affecting the wound healing process and every other process in the body.
Reepithelialization is a stage of healing that occurs during the proliferation phase and is regulated by growth factors. Measuring the level of re-epitheliazation is a frequently used method for the histopathological evaluation of the healing process.44,45 It was stated that the level of epithelialization in the tibia tissue of the rat group in which ABS was used in the treatment was higher than that in the control group.44 ABS is effective in full-thickness wound healing by accelerating proliferation in the epithelial tissues of rats.39 In a similar study, ABS, which was used for treating rat palatal mucosal injuries, increased the level of reepithelialization.50 Another similar study revealed that ABS contributed to epithelialization in a full-thickness skin wound model in rats.26 According to our results, the rate of re-epitheliazation was the highest in the ABS-dopped dressing group (III), and it was limited in the local ABS group (I) (Figure 5, Table 4). It was considered that brown scab formation, which was observed more prominently in the ABS-dopped dressing group than in the other groups, occurred because of reepithelialization and was caused by the formation of an encapsulated protein network on the wound area. Consistent with our results, scab formation was observed in the wound area in similar studies using ABS.51,52,53
Inflammation is a reaction developed by the body in cases of tissue damage, exposure to certain chemicals, and infectious diseases. This situation, in which the substances taking part in the immune system are directed to the damaged tissue, is an important parameter used in evaluating wound healing.26 While inflammation is not observed in the normal healing process, its severity may increase in necrotic conditions.53 In wound treatment, interventions that prevent inflammation are preferred. The use of ABS prevented inflammation and necrosis in experimentally created bone defects in the tibia of rats.45 It has been reported that ABS does not cause inflammation in experimentally induced full-thickness skin wound healing in rats.39 According to another study, ABS prevented inflammation by inhibiting collagen destruction during the recovery period.53,54 ABS reduced inflammation in a wound model created in rats.27 Likewise, it was reported that the use of ABS in rats with colonic anastomosis-induced collagen formation and increased anastomosis.55 In a study that was conducted to prevent postoperative intra-abdominal adhesions using ABS, an increase in inflammatory reactions to fibrosis was observed in the groups using ABS.49 In the evaluation made with the available data, it was determined that inflammation did not occur in any animal in our study, and this situation was compatible with most other studies in the literature.
OSI is a measure of oxidative stress. Oxidative stress occurs because of the overproduction of reactive oxygen species or when antioxidant protection is insufficient.56 It has been reported that increased OSI values in many conditions, such as diabetes, wounds, burns, and aging, can be reduced using the antioxidant properties of various herbal extracts.17,57,58,59,60,61,62,63,64,65,66,67 Our results showed that ABS, which is an herbal product, is effective against oxidative stress that occurs during the wound healing process, in line with the literature. The use of only dressing and ABS alone corresponded to a reduction in the increased stress by 1.5 times (p < 0.05). It was determined that the ABS-dopped dressing increased the TAS levels by approximately twice, while reducing the OSI values by 1/3 compared with the control (Table 5).
CONCLUSION
The results showed that ABS alone did not contribute to wound healing, and the ABS-dopped wound dressing provided rapid and uneventful healing in the injured area. It was determined that local ABS application did not shorten the wound healing duration, and the ABS-dopped nanofiber dressing provided healing in the injured area in a much shorter time than the control group. Because it does not cause inflammation, ABS is a hemostatic agent with antiseptic and antimicrobial properties. Our results indicate that ABS-dopped dressings can be used safely, do not have any negative effects on wound healing, and can be an effective and safe product for use by shortening the healing process in skin wounds.
Preparation of the nanofibers
In this study, nanofibers were prepared to form a homogeneous wound dressing surface and provide a longer-lasting effect by the slow release of the drug additive. In addition, a control group was formed to examine the effects of the nanofiber surface with and without the drug additive and show the effects of the drug. The electrowinning process was performed for nanofiber wound dressing production.12,18,19 Sterile gauze was used for fiber collection, and two different solutions were used as polymer solutions.9 First, 10% by mass PVA (Mw: 72.000) in pure water was obtained by stirring at 70 °C for 1 h. Then, 10% PVP (MW: 58.000) in ethanol was prepared using a magnetic stirrer at room temperature for 30 min. These two polymer solutions were mixed at room temperature at a ratio of 3/4 PVA and 1/4 PVP to obtain a carrier polymer solution. 10 mL of the carrier polymer solution was used to make nanofiber without any additive. Taking 10 mL of the same polymeric solution, 1 mL of Ankaferd was added and nanofibers were produced. The unhopped fiber was collected on sterile gauze with a rotary collector at a speed of 400 rpm under the influence of an electric field of 1.7 kV/cm at a flow rate of 1 mL/h. The ABS-doped fiber was collected with a rotary collector at a speed of 400 rpm under an electric field of 1.9 kV/cm at a flow rate of 0.6 mL/h.
Experiments on material characterization
The experiments in this context were conducted at Necmettin Erbakan University, Science and Technology Research and Application Center (BITAM).
SEM micrographs of polymers
SEM was used to examine the morphology of the produced materials. The surface morphologies of the polymers were examined at magnifications of 1.000 and 5.000.
FTIR analysis
FTIR analyses were performed to determine the interaction between the drug mixture and the polymer structure.20
Animal materials and treatments
Twelve-week-old female Wistar albino rats with an average weight of approximately 295 g were used in this study. Female rats were preferred because the skin of females is thinner than that of males, and the lack of estrogen in males would negatively affect cutaneous wound healing.21,22 The rats were housed without any water or feed restriction at room temperature in a 24 hours light-dark cycle.23 The study was planned to include 24 rats, with four groups (n= 6 each) (Table 1).
After the groups were formed, the rats were administered 70 mg/kg ketamine and 10 mg/kg xylazine through the intraperitoneal (i.p.) route.24,25 In each rat whose back area was shaved, a wound with a diameter of 15 mm was created using a biopsy needle. Group I was not treated. Group II received a single dose of local ABS in spray form. ABS-dopped wound dressing was used for group III, non-dopped ABS wound dressing was used for group IV (Figure 1), and the study was completed on day 14.26
Histological evaluation
The skin tissue samples were embedded in paraffin after routine tissue processing steps, and the samples were fixated in 10% formaldehyde for histological analyses.27 Sections of paraffin blocks with thicknesses of 6 µm each were stained with hematoxylin-eosin to determine their general histological structures.28 The sections were examined at 40x magnification under a light microscope equipped with a digital camera (Nikon Eclipse, E-400 equipped with Nikon DS Camera Control Unit DS-L1 with DS Camera Head DS-5M), and digital images of the relevant areas were taken.29,30,31,32
Scoring evaluation scale
Wound scoring was performed as 0 (no inflammation), 1 (mild inflammation), 2 (moderate inflammation), and 3 (severe inflammation). Wound diameter was recorded in millimeters.25,26 The scoring process was performed according to the evaluation scale shown in Table 2.
Biochemical analysis
On the last day of the study, 1.5 mL of blood was intracardially taken from each rat. After the blood samples were centrifuged for 3000 cycles at +4 °C for 10 min, the serum parts were separated. TAS and TOS measurements of sera were performed using commercial kits (Rel Assay Diagnostics). Using a spectrophotometer (Biochrom Libra S22), the absorbances of the samples were measured at 660 nm, and OSI was determined according to the standard reference.33,34
Statistical analysis
The Statistical Package for the Social Sciences (SPSS, version 21.0, IBM Corporation, Armonk, NY) software was used for all statistical analyses. One-way analysis of variance and the least significant difference method were used for pair-wise comparisons (p < 0.05). Statistical analysis was performed on the 14th day in the scoring of wound diameters.
Materials and characterization
SEM micrographs of polymers
Using the SEM images, the average fiber diameters were calculated with the help of the “Fiji ImageJ for Windows V 1.8.0” program from National Institutes of Health images, and the plots shown in Figure 3 were obtained. The fiber diameters ranged from 400 nm to 2 µm. The average diameter of the unadulterated fibers was 1.066 µm, and the average diameter of the doped fibers was 0.974 µm. The determined average diameter values were sufficient to provide permeability.35,36,37 While a mesh structure was detected at the points where the fibers overlapped on the surfaces of the doped fibers, no droplet or clump-like formation was observed on the surfaces (Figure 2).
FTIR analysis
In the FTIR spectra of the PVA-PVP polymer mixture, an O-H stretching vibration peak was observed at approximately 3290 cm-1. The spectrum band corresponded to a C-H stress that occurred at a peak of asymmetrical stretching vibration of approximately 2862 cm-1.35,37 PVA and PVP had C=O groups showing a vibration band at about 1716 cm-1. The C-N stretching vibration peak of the amine structure was observed at 1242 cm-1. The spectrum bands for the C-O groups, which are acetyl groups, appeared at 1100 cm-1. The plane for the C-H bend was outside the rings and formed an absorption band of approximately 720 cm-1.36 In the case of the ABS-dopped fibers, a soft formation of around 1645 cm-1 was observed instead of the peak loss at 1716 cm-1. Here, the oxygen band in the C=O group was transformed into a C=C stretching vibration by a free reaction. It was concluded that an oxidation reaction occurred by the binding of oxygen atoms in the ABS additive (Figure 4).
Body weights
It was determined that there was no statistically significant difference between the mean body weights of the rats in the groups (p= 0.643). The statistics of the test were found to be F23= 0.497 (p > 0.05) (Table 3).
Wound scores
There was no loss of animals in the groups. In the evaluations of the median scores on the last day of the study, the average wound diameter was found to be reduced by half compared with the first day of the study in the rats in group I, whereas the recovery in group II was more limited. In group III, where the best healing process was observed, almost all wounds were closed, and there was even hair growth in the injured area. It was determined that the recovery rate in group IV was faster than that in groups I and II (Table 4). In the Tukey and Duncan tests, the test statistics were found to be p= 0.019, degree of freedom= 23, and F=4.144. When the wound diameters were compared, it was seen that the 1st and 4th groups were nearly similar. It was observed that the wound diameter in the second group did not decrease. It was determined that the wound diameter in the 3rd group decreased statistically significantly (wound diameter: group II > I > V > III; p < 0.05).
There was no significant difference among the groups in the macroscopic wound measurements performed on day 3 (Figure 5, p > 0.05). In the evaluation made on the seventh day, brown-colored scab formation was observed in all groups. The wound healing rate was the highest in group III, and the healing rate in group IV was higher than those in groups I and II (p < 0.05). In the histopathological evaluations, reepithelization was found to occur at a high rate in groups III and IV, at a moderate rate in group I, and at a minimum rate in group II (p < 0.05). On the 14th day, the wounds were completely closed in all rats in group III, whereas the wounds of the rats in group IV were substantially closed. The wounds were partially closed in group I, and healing was more limited in group II (Figure 5). In summary, reepithelization occurred at the maximum level in group III, at a high level in group IV, at a moderate level in group I, and at the minimum level in group II (reepithelization rate: group III > IV > I > II; p < 0.05).
In groups III and IV, the borders of the epidermis, dermis, and hypodermis showed normal morphological features, and the densities of connective tissue and collagen in the dermis layer were sufficient, whereas the density of collagen in the other groups was insufficient. Wound healing was characterized by a decrease in the number of neutrophils and new vascularization (Figure 6). The ABS-dopped wound dressing accelerated healing, whereas the non-dopped ABS wound dressing had a limited effect on wound healing (Figure 2). In addition, it was determined that the wound healing rate in the group that was administered local ABS to the injured area was slower than that in the control group.
Biochemical analysis
The level of oxidation (TOS) was the highest in the control group. ABS was found to reduce oxidation (Table 5). The application of wound dressing with the ABS additive caused a significant decrease in TOS values (Table 5). In parallel with these results, the level of antioxidants (TAS) increased in groups IV and II, but the highest increase was observed in group III (p < 0.05) (the OSI ranking: group III > IV > II > I).
